Research Article pubs.acs.org/acschemicalneuroscience
Mass Spectral Charting of Neuropeptidomic Expression in the Stomatogastric Ganglion at Multiple Developmental Stages of the Lobster Homarus americanus Xiaoyue Jiang,† Ruibing Chen,‡,∥ Junhua Wang,† Anita Metzler,§ Michael Tlusty,§ and Lingjun Li*,†,‡ †
School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, Wisconsin 53705-2222, United States Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, Wisconsin 53706-1396, United States § Lobster Research and Rearing Facility, Edgerton Research Laboratory, New England Aquarium, Central Wharf, Boston, Massachusetts 02110-3399, United States ∥ Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China ‡
S Supporting Information *
ABSTRACT: The stomatogastric nervous system (STNS) of the American lobster Homarus americanus serves as a useful model for studies of neuromodulatory substances such as peptides and their roles in the generation of rhythmic behaviors. As a central component of the STNS, the stomatogastric ganglion (STG) is rich in neuropeptides and contains welldefined networks of neurons, serving as an excellent model system to study the effect of neuropeptides on the maturation of neural circuits. Here, we utilize multiple mass spectrometry (MS)-based techniques to study the neuropeptide content and abundance in the STG tissue as related to the developmental stage of the animal. Capillary electrophoresis (CE)-MS was employed to unambiguously identify low abundance neuropeptide complements, which were not fully addressed using previous methods. In total, 35 neuropeptides from 7 different families were detected in the tissue samples. Notably, 10 neuropeptides have been reported for the first time in this study. In addition, we utilized a relative quantitation method to compare neuropeptidomic expression at different developmental stages and observed sequential appearance of several neuropeptides. Multiple isoforms within the same peptide family tend to show similar trends of changes in relative abundance during development. We also determined that the relative abundances of tachykinin peptides increase as the lobster grows, suggesting that the maturation of circuit output may be influenced by the change of neuromodulatory input into the STG. Collectively, this study expands our knowledge about neuropeptides in the crustacean STNS and provides useful information about neuropeptide expression in the maturation process. KEYWORDS: Homarus americanus, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI TOF/TOF MS), capillary electrophoresis (CE), neuropeptide, stomatogastric ganglion (STG), stomatogastric nervous system (STNS), development
T
he crustacean stomatogastric nervous system (STNS) has been a premier model system for the understanding of the generation of rhythmic motor patterns in circuit analysis for decades.1,2 This model preparation contains relatively large neurons that are easily identifiable, with the capability to produce motor patterns even after being removed from the animal. The neuronal circuits are extensively modulated by many substances, ranging from amine neurotransmitters to a large array of neuropeptides.1,3−6 These features of the STNS make it an attractive preparation to investigate the underlying mechanisms of central pattern generators and other circuits.6,7 Among various crustacean species, the American lobster Homarus americanus has become a particularly useful model system for the study of neural development. The lobster starts its life as an embryo, followed by 3 larval stages before the © 2012 American Chemical Society
metamorphosis into the postlarval stage (stage IV). Subsequent juvenile stages follow for several years before the lobster becomes an adult. Previous studies showed that the embryonic network could produce a single rhythmic pattern,8 but in a mature network (after mid larval stages), two distinct rhythms, the pyloric rhythm and the gastric mill rhythm, could be produced.8−10 This difference in functional output could be attributed to the immaturity of the synaptic connections, the neuromodulatory environment, or the membrane properties of the embryonic network. It has been reported that single Received: January 22, 2011 Accepted: March 1, 2012 Published: March 1, 2012 439
dx.doi.org/10.1021/cn200107v | ACS Chem. Neurosci. 2012, 3, 439−450
ACS Chemical Neuroscience
Research Article
Figure 1. Neuropeptides detected in (a) single adult STG by direct tissue analysis and (b) tissue extract of 15 pooled adult STGs using MALDI TOF/TOF MS. Peak assignment is based on accurate mass measurement and CID confirmation. While peptide profiles are very similar, minor differences are noted due to the removal of salts and additional sample preparation steps in tissue extraction. Unlabeled peaks are unidentified peaks. The right axis in (a) shows the absolute intensity of the spectrum. Mass spectra with entire mass range analyzed (m/z 600−2000) are provided in Supporting Information, Figure S1.
midembryonic development, but others do not appear until mid larval stages. Marder and co-workers used an electrophysiological approach to demonstrate that receptors for many neuromodulators appear and function before the motor patterns of the STNS are mature.14,18,19 These previous studies suggest the sequential recruitment of neuropeptides as a function of development. However, given the large number of isoforms of several peptide families, it remains unknown whether different individual members of a given peptide family appear at different developmental stages. To address this question, a methodology with the capability to detect multiple peptides simultaneously is needed. Mass spectrometry (MS) has evolved as a powerful tool for peptide analysis due to its high sensitivity, chemical specificity, speed, and capability for analyzing highly complex mixtures.
embryonic networks could generate adult-like outputs upon manipulation, indicating that the adult STG network backbone is already established by midembryonic development.11−13 In addition, the embryonic motor pattern could also be produced from adult preparation when certain neuromodulators were applied.13 Therefore, it is tempting to hypothesize that the immature output of the embryonic network could be largely attributed to the neuromodulatory environment and that the neuropeptide complement changes as a function of the neural network development. To study the neuropeptides in the STNS, extensive research has been carried out with a variety of approaches. Several studies using immunocytochemistry indicated that neuropeptides are acquired sequentially in the STNS network.14−17 Some neuromodulators are present in the STNS as early as 440
dx.doi.org/10.1021/cn200107v | ACS Chem. Neurosci. 2012, 3, 439−450
ACS Chemical Neuroscience
Research Article
Table 1. Neuropeptides Detected in the STG of Adult American Lobster Homarus americanusa [M + H]+ peptide family AST-A type
AST-B type FaRPs
orcokinin
proctolin SIFamide tachykinin
others
sequence
theoretical
observed
error (ppm)
RQYAFGLamide PRNYAFGLamide PRDYAFGLamide TNWNKFQGSWamide STNWSSLRSAWamide GGRNFLRFamide SGRNFLRFamide GNRNFLRFamide GDRNFLRFamide GPPSLRLRFamide APSKNFLRFamide GAHKNYLRFamide SMPSLRLRFamide DQNRNFLRFamide pQDLDHVFLRFamide DTSTPALRLRFamide QDLDHVFLRFamide GYSDRNYLRFamide FSHDRNFLRFamide FDAFTTGFGHN VYGPRDIANLY SSEDMDRLGFGFN NFDEIDRSGFGFV NFDEIDRSGFGFN NFDEIDRSGFGFH NFDEIDRSGFG NFDEIDRSGFA NFDEIDRSGFGF RYLPT VYRKPPFNGSIFamide APSGFLGMRamide APSGFLGM(O)Ramide TPSGFLGMRamide HI/LASLYKPR HIGSLYRamide
853.47 936.51 937.49 1266.64 1293.63 965.54 995.55 1022.56 1023.55 1041.63 1078.62 1104.61 1105.63 1208.63 1271.68 1275.72 1288.68 1289.64 1337.67 1213.53 1280.66 1474.63 1502.69 1517.67 1540.68 1256.55 1270.57 1403.62 649.37 1423.78 934.49 950.49 964.5 1084.62 844.48
853.47 936.5 937.5 1266.63 1293.64 965.54 995.52 1022.57 1023.56 1041.63 1078.62 1104.61 1105.63 1208.62 1271.64 1275.71 1288.64 1289.63 1337.69 1213.54 1280.66 1474.65 1502.69 1517.66 1540.68 1256.57 1270.6 1403.63 649.37 1423.78 934.49 950.49 964.51 1084.62 844.48
−3.3 −7.66 8.82 −3.97 5.13 −4.15 −30.89 5.57 7.24 0.4 −4.04 −4.29 −3.34 −9.86 −28.66 −5.76 −29.92 −6.79 11.44 12.13 2.73 10.19 0.99 −4.26 −1.66 13.09 23.61 7.3 −1.35 1.91 1.34 5.1 11.5 2.12 −2.49
MS/ MS xb xb x x
x x x
x x x
x x x x x x
x x x x
x x
CE fractions
ESTs identified
25 22, 23 20, 21 22, 23 24, 25, 26 33, 34 32, 33 33 30 31, 32, 33, 34 31, 32 30, 31 31, 32, 33, 34 25, 26, 27 17 22, 23, 24, 25 27 28 N/A N/A 16, 17 N/A 16, 17 16 17, 18 N/A 17 16 22, 23, 24, 25 27, 28, 29, 30 23, 24, 25, 26, 27 23, 24, 25, 26 23, 24, 25, 26 32, 33 27, 28
− − − − − − − − − − − − − − + − + − − + + + + + + − − − − + + + − − -
accession no.
BLAST score
Evalue
FD699285
163
6E-10
FD699285
163
6E-10
DV774522c DV774081c FC071459c DV772231c DV771438c DV774848c
171 198 197 143 170 197
5E-07 5E-07 5E-07 6E-07 6E-07 6E-07
CN952058 EX567979 EX567979
235 373 373
1E-18 3E-34 3E-34
a
AST, allatostatin; FaRPs, FMRFamide-related peptides. Others are peptides that do not fall into any known peptide families. Previously known peptides are shown in normal font; peptides previously reported in other species or other organs of the lobster, but observed in the STG for the first time, are shown in bold. Neuropeptides with similar sequences have similar retention time. CE elution order of the neuropeptides in lobster STG under negative mode was pGlu modified > orcokinin > AST-A > AST-B > proctolin > tachykinin > high mass (m/z > 1200) FaRPs > SIFamide > low mass (m/z < 1200) FaRPs. Notably, the overlaps of isoforms in different peptide families are expected. bTandem MS was performed together with the isotopic peak of CabTRP Ia. cFDAFTTGFGHN, VYGPRDIANLY, SSEDMDRLGFGFN, NFDEIDRSGFGFV, NFDEIDRSGFGFN, and NFDEIDRSGFGFH can all be found in these six ESTs.
focused on relatively high abundance peptides in the adult nervous system, and the comparative peptide analysis in adult and embryonic preparations only examined two conditions. The characterization of lower abundance peptides with relative quantitation across multiple developmental stages has not been addressed yet. Capillary electrophoresis (CE) provides a powerful tool to alleviate extreme chemical complexity of tissue extracts by overcoming analyte suppression and the limited dynamic range of MS analysis. We demonstrated that the off-line coupling of CE to MALDI MS analysis enabled global analysis of neuropeptides with enhanced peptide detection using even a 10-fold less sample amount as compared to direct MALDI MS analysis.39,40 The current study expands upon previous investigations on neuropeptide identification by characterizing low abundance neuropeptides with the incorporation of CE
The last several years have witnessed explosive discovery and elucidation of neuropeptides in the crustacean nervous system using MS-based approaches.17,20−32 With the advent of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), it became possible to detect biological species with molecular specificity in very limited samples such as single organs or single cells.33,34 Several sample preparation methods employing either direct tissue analysis or pooled tissue extraction have proven to be effective for neuropeptide analysis.25,35 We have previously identified a total of 102 peptides in the nervous system and neuroendocrine organs of Homarus americanus using a combination of direct tissue and pooled extract analysis.36,37 Furthermore, both methods have been employed to compare the neuropeptide complement of the STG and brain in the adult and embryonic lobsters.38 However, previous peptidomic studies mainly 441
dx.doi.org/10.1021/cn200107v | ACS Chem. Neurosci. 2012, 3, 439−450
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Figure 2. Collisional induced dissociation (CID) mass spectra of neuropeptides from the adult STG of the lobster H. americanus acquired on a MALDI TOF/TOF mass spectrometer. (a) CabTRP Ia, APSGFLGMRamide (m/z 934.49); (b) orcokinin, NFDEIDRSGFGFV (m/z 1502.69); (c) other peptide, HIGSLYRamide (m/z 844.48). All precursor ions are singly charged. The sequence-specific b- and y-type fragment ions and immonium ions are labeled.
■
separation prior to MS analysis to improve peptidome coverage. Overall, 35 neuropeptides from seven different families were detected in the STG, including 10 neuropeptides reported for the first time in this study. A more complete neuropeptide list would provide a useful guide for further functional studies that would lead to improved mechanistic insight into the regulatory role of these signaling peptides in neural network development. In addition, the neuropeptidomic expression patterns at different developmental stages were compared and sequential appearance of neuropeptides was also highlighted. Collectively, this work employed analytical tools to generate a more complete STG peptidomic profile and presented a relative quantitation method to survey STG peptide expression at multiple developmental stages. These results will lead to an improved understanding of neuropeptide regulation in the maturation process at the network level.
RESULTS AND DISCUSSION
The stomatogastric ganglion (STG) connects to the central nervous system (CNS) via a single input nerve (the stomatogatric nerve, stn) and a single output nerve to control motor movements of the stomach. Despite size differences, the general morphology of the STG and the number of neurons within the STG remain the same throughout developmental stages, thus yielding similar peptide extraction efficiencies. The discovery and identification of neuropeptides in the STG of H. americanus via mass spectrometry (MS) has been described in several previous reports.17,36,38,41 However, due to the chemical complexity and small tissue size, the majority of previous studies used only accurate mass matching method for the identification of relatively high-abundance neuropeptides. The identification and assignment of neuropeptides in low abundance were proven to be difficult. Here, we utilized a combination of direct tissue analysis, tissue extraction profiling, 442
dx.doi.org/10.1021/cn200107v | ACS Chem. Neurosci. 2012, 3, 439−450
ACS Chemical Neuroscience
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Figure 3. MALDI TOF/TOF mass spectra of (a) the adult STG extract and (b−e) CE separation of three peaks APSGFLGMRamide (m/z 934.49), PRNYAFGLamide (m/z 936.51), and PRDYAFGLamide (m/z 937.49). (a) Two peaks m/z 936.51 and m/z 937.49 overlap with the isotopic cluster envelope of peak m/z 934.49 in the tissue extract profiling. After CE fractionation, these three peaks are separated, with (b) m/z 937.49 present only in fraction #21, (c) m/z 936.51 in fraction #22, (d) both m/z 934.49 and m/z 936.51 in fraction #23, and (e) m/z 934.49 only in fraction #24. The appearances of isolated peaks at m/z 936.51 and m/z 937.49 in distinct fractions confirm the existence of these two peptides.
high confidence for neuropeptide identification. Figure 2b and c shows MS/MS spectra of lower abundance peptide peaks, an orcokinin peptide NFDEIDRSGFGFV (m/z 1502.69), and a peptide not belonging to any known family, HIGSLYRamide (m/z 844.48). The majority of sequence-specific fragment ions are observed, offering solid confirmation for peptide peak assignment. Comparisons with Previous Studies on Adult STG by MS. The MALDI TOF/TOF analysis indicated that Val1SIFamide (m/z 1423.78 VYRKPPFNGSIFamide) was the highest peak in the mass spectra, which is in accordance with a previous study of adult STG by MALDI FTMS.41 It appears to be the sole isoform in the SIFamide family in the lobster, H. americanus. Immunocytochemical and physiological studies have demonstrated its widespread distribution and local neuromodulatory role throughout the STNS.41 Nonetheless, another previous analysis of the adult lobster STG using high pressure MALDI FTMS revealed that [Asn13]orcokinin (m/z 1517.67 NFDEIDRSGFGFN) is the most abundant peak in a majority of the mass spectra.38 To address this difference, we further tested the adult STG on high pressure MALDI FTMS and observed a result very similar to that of MALDI TOF/TOF with Val1-SIFamide being the highest peak (Figure S2, Supporting Information). This discrepancy might be due to the different batches of animals, different environmental conditions, or different parameters of the instrument. In crustaceans, SIFamide might play a role in processing or transmitting tactile, olfactory, and visual stimuli.42 The highest signal response in MALDI MS measurement also might be due to the presence of multiple basic residues in its sequence, and therefore, further investigations would be necessary to establish the absolute concentrations of these peptides. CE-MALDI for Improved Neuropeptide Characterization. In addition to mass matching and tandem mass fragmentation, a microscale separation technique, CE was also incorporated prior to MS detection. With CE, interfering components could be eliminated, and closely spaced peaks in
sequence-specific tandem MS fragmentation, and CE separation of tissue extract to unambiguously assign several lowintensity peaks in the mass spectra. Furthermore, the quantification study mapped the neuropeptide changes at different stages and demonstrated similar trends of isoform level changes within one family. This integrated methodology enabled a more in-depth characterization of neuropeptidome in developing H. americanus STG. Neuropeptide Assignment and Identification by MALDI TOF/TOF. Figure 1 shows representative mass spectra of the adult STG acquired using direct tissue and tissue extraction analyses. Tissue extraction of 15 STGs (Figure 1b) produces a cleaner spectrum, presumably due to a combination of larger peptide amounts and a Ziptip cleanup procedure, whereas direct tissue analysis of the STG produces more noise in the low mass range (Figure 1a), possibly due to the limited sample amount (1 single tissue) and incomplete salt removal. However, the direct tissue method is superior to tissue extraction, considering its speed and simplicity of sample preparation. It only requires mashing of the sample to expose neuropeptides to the surface and then applying acidic dihydroxybenzoic acid (DHB) to extract and crystallize the analytes. Both methods generated spectra with similar peptide patterns, including relative abundances, indicating relatively stable neuropeptide expression levels and the feasibility of both methods (Figure 1 and Figure S1 (Supporting Information)). The peaks were first assigned based on accurate mass measurements by matching with calculated masses from previous reports36,38 and an in-house H. americanus neuropeptide database. The mass measurement accuracy for assignment was set at 30 ppm. Most of the peptide identities were also confirmed by collisional-induced dissociation (CID) fragmentation performed directly on tissue or tissue extract, as indicated in Table 1. Figure 2a shows a representative tandem mass spectrum for Cancer borealis tachykinin related peptide (CabTRP) Ia APSGFLGMRamide (m/z 934.49). As seen, an almost complete peptide sequence can be obtained, providing 443
dx.doi.org/10.1021/cn200107v | ACS Chem. Neurosci. 2012, 3, 439−450
ACS Chemical Neuroscience
Research Article
Figure 4. MALDI TOF/TOF mass spectra of the adult STG. (a,b) Direct tissue profiling and extraction profiling of Val1-SIFamide VYRKPPFNGSIFamide (m/z 1423.78), its sodium adduct peak (m/z 1445.76), and potassium adduct peak (m/z 1461.87), with higher salt adduct intensities in direct tissue analysis. (c,d) After CE separation, the peaks with larger masses are coeluted with m/z 1423.78 in fractions #27 and #28, confirming their identities of salt adducts of Val1-SIFamide.
As shown in Figure S3 (Supporting Information), peaks at m/z 950.5 and 964.5 are better resolved after coeluting with the other tachykinin neuropeptide CabTRP Ia (m/z 934.49) with a much improved intensity. All tachykinins are eluted much earlier than the peak m/z 965.54 from the FaRP family, so the ambiguity is eliminated. More detailed mapping of peptides and their CE migration time is shown in Table 1. Another example of utilizing CE to assist peptide identification is to assign sodium and potassium adduct peaks based on CE profiles. In direct MS profiling experiments, we detect both Val1-SIFamide VYRKPPFNGSIFamide (m/z 1423.78) and two additional peaks at m/z 1445.8 and 1461.8 in the STG of lobster. Notably, the relative intensities of these two peaks are higher in the direct tissue analysis than tissue extract detection (Figure 4a and b), with the first method bearing more salts than the second one. After CE separation, we observe that the peaks m/z 1445.8 and 1461.8 always appear together with Val1-SIFamide (Figure 4c and d), as in fractions #27 and #28, suggesting that these two peaks are likely Na+ and K+ adducts. These results are also in agreement with the accurate mass measurement result (
